KR20010072888A - Drugs for relieving carbonyl stress and peritoneal dialysates - Google Patents

Abstract

An intraperitoneal carbonyl stress improving agent in a peritoneal dialysis solution containing a carbonyl compound trapping agent as an active ingredient. Carbonyl compound trapping agents, such as aminoguanidine, inactivate or remove the carbonyl compound produced and accumulated in the peritoneal dialysis solution. The carbonyl compound produced during the liquefaction bactericidal and preservation of the peritoneal dialysis solution is removed by contacting the trapping agent in advance. In addition, by adding a trapping agent to the peritoneal dialysis solution or circulating using a carbonyl compound trap cartridge, the carbonyl compound derived from the patient's blood, which is accompanied by the peritoneal dialysis and flows out into the abdominal cavity, can also be removed. According to the present invention, modification of the intraperitoneal protein by the carbonyl compound can be suppressed, and the peritoneal injury accompanying peritoneal dialysis can be reduced.

Description

Drugs for relieving carbonyl stress and peritoneal dialysates

Dialysis performed in patients with chronic renal failure includes hemodialysis and peritoneal dialysis. Peritoneal dialysis is a dialysis method performed by axial flow of a dialysate in a peritoneal cavity for a predetermined time, discharging waste products in the body into the dialysate through the peritoneum, and then recovering the dialysate. Peritoneal dialysis is largely divided into intermittent peritoneal dialysis (IPD) and continuous ambulatory peritoneal dialysis (CAPD). The CAPD method is a peritoneal dialysis method which introduces the advantages of the IPD method, lengthens the retention time of the perfusion liquid injected into the abdominal cavity, and makes liquid exchange about four times a day.

Peritoneal dialysis has advantages such as simple and low time constraints. However, it is known that deterioration of hydration capacity gradually decreases after peritoneal dialysis for a long period of time, resulting in degeneration of the abdominal protein, curing, and peritoneal fusion.

Some of these causes are considered to be in glucose contained in the peritoneal dialysis solution. Currently, most of the peritoneal dialysis solution used contains glucose as a penetration regulator. Glucose is considered to be unstable to heat, and partly decomposes during sterilization, so that a highly reactive carbonyl compound capable of modifying the protein is produced as a degradation product. In addition, peritoneal dialysis solution containing glucose is considered to produce and accumulate decomposition products during storage after sterilization.

In general, since the decomposition of glucose is likely to occur from the neutral side to the basic side, in general, peritoneal dialysis solution, in consideration of the stability of glucose, a buffer system which gives a pH of the acid side (pH 5.0 to 5.4) is often used. . However, such acid peritoneal dialysis solution is concerned about the deterioration of the immune defense mechanism of the abdominal macrophages, the occurrence of peritonitis due to the entry of bacteria, and the injury to peritoneal mesothelial cells. In order to solve these conflicting problems, it is urgently desired to prevent the production of carbonyl compounds or to remove the carbonyl compounds resulting from the decomposition of glucose in the peritoneal dialysis solution in the neutral vicinity.

On the other hand, from the viewpoint that the peritoneal dialysis solution containing high concentration of glucose is not preferable in the peritoneum, such as modifying the protein, a peritoneal dialysis solution using a glucose polymer having a lower decomposition product is being developed (Japanese Patent Application Laid-Open No. 10-94598). Wilkie, ME et al., Perit.Dial.Int., 17: S47-50 (1997).

At the same time, cyclodextrins (JP 8-71146), disaccharides (JP 8-131541), amino acids (Faller, B. et al., Kidney Int., 50 (suppl) were substituted for glucose as a penetration regulator. 56, peritoneal dialysis solution using S81-85 (1996)). Moreover, the peritoneal dialysis solution which suppressed the degradation of glucose by adding cysteine is also disclosed (Japanese Patent Laid-Open No. 5-105633).

These methods are all aimed at improving the problems caused by high concentration of glucose in peritoneal dialysis solution.

However, in chronic renal failure patients, highly reactive carbonyl compounds and AGE (Advanced glycation end products) have been reported to accumulate in blood and tissues regardless of the presence of hyperglycemia (Miyata, T. et al., Kidney Int., 51: 1170-1181 (1997), Miyata, T. et al., J. Am. Soc. Nephrol., 7: 1198-1206 (1996), Miyata, T. et al., Kidney Int. 54: 1290-1295 (1998), Miyata, T. et al., J. Am. Soc.Nefrol. 9: 2349-2356 (1998). In renal failure, a condition in which a carbonyl compound becomes a high load state (carbonyl stress) by a non-enzymatic biochemical reaction and protein modification is enhanced, and a carbonyl compound is produced from sugar and lipids to modify the protein. (Miyata, T. et al., Kidney Int. 55: 389-399, (1999)). Carbonyl stress is not only a problem of changing the construction of matrix proteins such as collagen and fibronectin, but also related to peritoneal permeability and inflammation due to the physiological activity of various cells of the carbonyl compound. It is thought to be.

In the case of peritoneal dialysis, waste products in the blood are excreted in the peritoneal dialysis fluid through the peritoneum. The high-permeability peritoneal dialysis fluid has a function of collecting highly reactive carbonyl compounds accumulated in blood of renal failure patients in the peritoneal dialysis fluid across the peritoneum. Therefore, a carbonyl stress state with a high carbonyl compound concentration in the peritoneal dialysis solution is caused, the protein in the peritoneum receives carbonyl modification, and the function of the peritoneum decreases, which is thought to be involved in the development of peritoneal sclerosis.

Indeed, in patients with peritoneal dialysis, the presence of carbonyl stress due to intraperitoneal glucose has been demonstrated from immunohistochemical examination of endothelial and mesothelial tissues (Yamada, K. et al., Clin. Nephrol., 42 : 354-361 (1994), Nakayama, M. et al., Kidney Int., 51: 182-186 (1997), Miyata, T. et al., J. Am. Soc.Nefrol. In press, Combet, S. et al., J. Am. Soc. Nephrol. In press, Inagi, R. et al., J. Am. Soc. Nephrol. In press).

The present invention relates to peritoneal dialysis solution for use in the treatment of patients with renal failure.

1 is a diagram showing the amount of carbonyl compound in peritoneal dialysis solution and peritoneal dialysis drainage liquid.

Fig. 2 is a photograph (top) and a schematic diagram (bottom) showing the histological localization of carbonyl modified protein in the peritoneum of peritoneal dialysis fluid patients. In the meantime, A represents a positive location in the connective tissue under the mesothelial cells which have been separated. B represents the benign site of the thickened blood vessel wall.

Fig. 3 shows the expression of VEGF mRNA in mesothelial cells to which glyoxal, methylglyoxal and 3-deoxyglycosone were added. Reverse transcription of all RNAs extracted from rat mesothelial cell cultures incubated with glyoxal (A), 3-deoxyglycosone (B) and methylglyoxal (C) at various concentrations (0, 100, 200, 400 μM) was performed. VEGF and G3PDH cDNA were amplified by 30 and 21 cycles of PCR, respectively. Three consecutive experiments were performed to obtain an average. For each experimental mean value, the ratio of VEGF mRNA to G3PDH mRNA was calculated. The experiment was performed three times and the average ± S.D was graphed. ※ P <0.0005.

Fig. 4 shows the production of VEGF protein in microvascular endothelial cells by methylglyoxal addition. Human microvascular endothelial cells were cultured in the presence of various concentrations of methylglyoxal (0, 200, 400 μM), and VEGF protein released into the culture supernatant was quantified by ELISA. Representative results of three experiments are shown. Data are expressed as mean ± range. ※ P <0.05, ※※ P <0.01.

Fig. 5 shows the expression of VEGF mRNA in endothelial cells to which methylglyoxal was added. Human endothelial cells were cultured in the presence of methylglyoxal at various concentrations (0, 100, 200, 400 μM), and total mRNA was extracted and reverse transcription was performed. VEGF and G3PDH cDNA were amplified by 30 and 21 cycles of PCR, respectively. The experiment was conducted three times in succession to find an average. For each experimental mean value, the ratio of VEGF mRNA to G3PDH mRNA was calculated. The experiment was performed three times and the average ± S.D was graphed. ※ P <0.05, ※※ P <0.005, ※※※ P <0.0001.

Figure 6 shows the expression of VEGF mRNA in rat peritoneal tissues intraperitoneally administered with methylglyoxal daily for 10 days. Expression of VEGF and G3PDH mRNA in the peritoneum was amplified by 28 and 16 cycles of RT-PCR, respectively. The experiment was conducted three times in succession to find an average. For each experimental mean value, the ratio of VEGF mRNA to G3PDH mRNA was calculated. The experiment was performed three times and the average ± S.D was graphed. ※※ P <0.05.

Figure 7 shows the carbonyl stress in the abdominal cavity of peritoneal dialysis patients.

FIG. 8 is a diagram showing the effect of addition of aminoguanidine to pentosidine production by intraperitoneal dialysis drainage incubation. FIG.

9 is a diagram showing the effect of the addition of aminoguanidine on the production of protein carbonyl by peritoneal dialysis drainage incubation.

Fig. 10 shows the effect of addition of aminoguanidine to the amount of carbonyl compounds in peritoneal dialysis (CAPD) fluid of three peritoneal dialysis patients (Patient I, Patient S, and Patient K).

Fig. 11 shows the effect of addition of aminoguanidine to 5-HFM production in peritoneal dialysis solution in an acidic environment.

Fig. 12 shows the effect of addition of aminoguanidine to 5-HFM production in peritoneal dialysis solution in neutral environment.

Fig. 13 shows the effect of adding aminoguanidine to the carbonyl compound in the peritoneal dialysis solution in acidic and neutral environments.

14 is a diagram showing the effect of inhibiting pentosidine production by adding carbonyl compound trapping beads to peritoneal dialysis solution.

Fig. 15 shows the effect of removing carbonyl compounds by addition of carbonyl compound trap beads to peritoneal dialysis solution.

Fig. 16 shows the effect of removing carbonyl compounds by the addition of activated carbon to the dicarbonyl solution.

Fig. 17 shows the effect of removing carbonyl compounds by the addition of activated carbon to the peritoneal dialysis solution.

Fig. 18 is a diagram showing the trapping effect on glyoxal, methylglyoxal and 3-deoxyglucosone when guanidine is used.

Fig. 19 is a diagram showing a trapping effect on glyoxal, methylglyoxal and 3-deoxyglucosone when metformin is used for biguanide.

Fig. 20 is a diagram showing a trapping effect on glyoxal, methylglyoxal and 3-deoxyglucoson when buformin is used for biguanide.

Fig. 21 is a diagram showing the trapping effect on glyoxal, methylglyoxal and 3-deoxyglucoson when phenformin is used for biguanide.

Fig. 22 is a diagram showing the trapping effect on glyoxal, methylglyoxal and 3-deoxyglucosone when aminoguanidine is used.

Fig. 23 shows the trapping effect on glyoxal, methylglyoxal, and 3-deoxyglucosone when cysteine is used for the SH compound.

Fig. 24 shows the trapping effect on glyoxal, methylglyoxal, and 3-deoxyglucosone when N-acetylcysteine is used for the SH compound.

Fig. 25 is a diagram showing the trapping effect on glyoxal, methylglyoxal and 3-deoxyglucosone when GSH is used for the SH compound.

Fig. 26 shows the trapping effect on glyoxal, methylglyoxal, and 3-deoxyglucosone when albumin is used as the SH compound.

Fig. 27 is a diagram showing the effect of inhibiting the production of pentosidine when the SH compound is added to the peritoneal dialysis solution.

Disclosure of the Invention

An object of the present invention is to provide a method for improving the disorder caused by a carbonyl compound in peritoneal dialysis, that is, a carbonyl stress state, and a dialysate or drug for realizing the method. The carbonyl compound in the present invention includes a carbonyl compound derived from a patient undergoing peritoneal dialysis, a carbonyl compound produced in the peritoneal dialysis solution itself or during its storage, and a carbonyl compound produced in the peritoneal cavity during peritoneal dialysis. It becomes a target. It is a subject of the present invention to make the disorder to dialysis patients by these carbonyl compounds as small as possible.

The inventors found out that the highly reactive carbonyl compound contained in the peritoneal dialysis solution injected into the peritoneal cavity of the patient does not originate from the peritoneal dialysis solution. That is, the carbonyl compounds other than glucose in the peritoneal dialysis drainage collected from the peritoneal dialysis patient were five times before dialysis, and the increase was considered to be a carbonyl compound derived from blood (Fig. 1). From this fact, the carbonyl compound in the peritoneal dialysis solution in addition to the carbonyl compound produced during the heat sterilization process of the peritoneal dialysis solution, or the carbonyl compound produced and accumulated during the storage of the peritoneal dialysis solution, the carbonyl compound derived from blood and It was also found that the presence of carbonyl compounds produced and accumulated in the abdominal cavity could not be ignored. Indeed, even in the examination of peritoneal tissues of peritoneal dialysis patients using immunostaining, carbonyl modified proteins were localized in the tissues (Fig. 2). Therefore, if the carbonyl compound flowing out of the abdominal cavity from the blood in conjunction with peritoneal dialysis can be removed, the carbonyl stress state can be improved more effectively.

The present inventors have a condition that promotes in vivo protein modification in renal failure, and in the case of continuously injecting high concentrations of glucose into the peritoneum like peritoneal dialysis, the peritoneal dialysis solution in which the carbonyl compound in the peritoneum is accumulated Thought to be placed in a state that is more susceptible to modification (figure 7).

Based on the above background, the present inventors have found that the carbonyl compound trapping agent is effective for producing a dialysis solution for reducing the carbonyl compound derived from the peritoneal dialysis solution, and completed the present invention. Furthermore, the present inventors focused on carbonyl compounds accumulated in the blood, and found a medicament capable of inhibiting peritoneal dialysis complications based on protein modification caused by carbonyl stress and completed the present invention.

That is, the present invention relates to a carbonyl stress state improving agent, a peritoneal dialysis solution and a drug applied thereto, and more specifically,

(1) intraperitoneal carbonyl stress state improving agent in peritoneal dialysis containing carbonyl compound trapping agent as an active ingredient,

(2) The carbonyl stress state improving agent according to (1), wherein the carbonyl compound trapping agent is immobilized on an insoluble carrier,

(3) The carbonyl stress state improving agent according to (1), wherein the carbonyl compound trapping agent is for incorporation into the peritoneal dialysis solution,

(4) The carbonyl compound according to any one of (1) to (3), wherein the carbonyl compound trapping agent is a compound selected from the group consisting of aminoguanidine, pyridoxamine, hydrazine, SH group-containing compounds, or derivatives thereof Stress improvers,

(5) The carbonyl stress state improving agent according to any one of (1) to (3), wherein the carbonyl compound trapping agent is a Maillard reaction inhibitor.

(6) The carbonyl stress state improving agent according to (1), wherein the carbonyl compound trapping agent is an insoluble compound in a peritoneal dialysis solution capable of adsorbing a carbonyl compound,

(7) a cartridge for trapping carbonyl compounds in a peritoneal dialysis solution filled with the carbonyl compound trapping agent of (2) and / or (6),

(8) a method for preparing peritoneal dialysis solution with reduced carbonyl compound content, comprising passing the peritoneal dialysis solution to the carbonyl compound trap cartridge of (7),

(9) a method for preparing a peritoneal dialysis solution with reduced carbonyl compound content, comprising the following steps:

a) contacting the peritoneal dialysis solution with the carbonyl compound trapping agent of (2) and / or (6), and

b) separating the carbonyl compound trapping agent and the peritoneal dialysis solution

(10) peritoneal dialysis solution containing a carbonyl compound trapping agent,

(11) The peritoneal dialysis solution according to (10), wherein the peritoneal dialysis solution contained in the divided chambers of the first chamber and the second chamber, wherein the reducing sugar is contained in the first chamber and the carbonyl compound trapping agent is contained in the second chamber. ,

(12) The peritoneal dialysis solution according to (10), wherein the carbonyl compound trapping agent is administered intraperitoneally with the peritoneal dialysis solution,

It is about.

The present invention also relates to the use of a carbonyl compound trapping agent in a method for improving the carbonyl stress state in the abdominal cavity. The present invention relates to the use of a carbonyl compound trapping agent in peritoneal dialysis treatment. Moreover, this invention relates to the use of the carbonyl compound trapping agent in the manufacturing method of a carbonyl stress improvement agent.

In this invention, the carbonyl compound used as a trap object is a carbonyl compound produced | generated during the manufacturing process and storage of a peritoneal dialysis solution, for example. As described above, the peritoneal dialysis solution containing high concentration of glucose as the penetration control agent is always accompanied by the possibility of producing carbonyl compounds. As this kind of carbonyl compound, the following substances are known, for example (Richard, J. U. et al., Fund. Appl. Toxic., 4: 843-853 (1984)).

3-deoxyglucoson

5-hydroxymethylfurfural (5-hydroxymethylfurfural, hereinafter abbreviated as 5-HMF)

Formaldehyde

Acetaldehyde

Glyoxal

Methylglyoxal

· Lefoic acid

Furfural

Arabinos

In the present invention, by using the carbonyl compound trapping agent during dialysis, not only the carbonyl compound produced during the preparation or preservation of the peritoneal dialysis fluid, but also accumulated in the blood of the renal failure patient and transported into the peritoneal cavity due to peritoneal dialysis Removal of the following carbonyl compounds can also be achieved.

Carbonyl Compounds Derived from Ascorbic Acid:

Dehydroascorbic acid

Carbonyl compounds derived from carbohydrates, lipids, or amino acids:

Glyoxal

Methylglyoxal,

3-deoxyglucoson

Hydroxy nonenal

Malondialdehyde

Acrolein

As the carbonyl compound trapping agent in the present invention, it is preferable that all of these carbonyl compounds lose or modify the carbonyl compound's hydrophilic activity against proteins by chemical reaction or adsorption. Also included are those that are effective only for the major of the compounds. For example, methylglyoxal has relatively high reactivity among carbonyl compounds (Thornalley, RJ, Endocrinol. Metab. 3: 149-166 (1996), Inagi, R. et al., J. Am. Soc). Nephrol. In press and Example 3), the activity of the deprivation is significant pathophysiological significance. Therefore, the compound effective against methylglyoxal can be said to be preferable as a carbonyl compound trapping agent in this invention. Specifically, as shown in the examples, compounds such as activated carbon, guanidine, aminoguanidine, beguanide, cysteine, and albumin are particularly effective carbonyl compound trapping agents for methylglyoxal.

Carbohydrates used as penetration pressure regulators have been reported to have better stability than glucose, but it is not easy to completely suppress the production of these carbonyl compounds during heat sterilization or storage. Therefore, even when using carbohydrates other than glucose, the use of the carbonyl compound trapping agent is meaningful. Examples of carbohydrates that can be used as a permeability regulator for peritoneal dialysis in addition to glucose include trehalose (JP-A-7-323084), hydrolyzed starch (JP-A-8-85701), maltitol and lactitol (extraordinary). 8-131541), or non-reducing oligosaccharides, non-reducing polysaccharides (Japanese Patent Application Laid-Open No. Hei 10-94598) and the like are known.

As the carbonyl compound trapping agent in the present invention, a compound which loses the damaging activity of a carbonyl compound to a dialysis patient by a chemical reaction or adsorption with a carbonyl compound, or a compound that lowers the compound itself. Use materials that are safe for dialysis patients. As such a compound, the following are known, for example. The carbonyl compound trapping agent of the present invention may be used alone or in combination of two or more kinds thereof.

Aminoguanidine (Foote, E. F. et al., Am. J. Kidney Dis., 25: 420-425 (1995)).

± 2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (± 2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide: OPB-9195) (S. Nakamura, 1997 , Diabetes. 46: 895-899)

Moreover, as a carbonyl compound trapping agent, the compound which functions as a carbonyl compound trapping agent can be used, for example as the following compounds or derivatives thereof. In addition, a derivative refers to the compound in which substitution of an atom or a molecule | numerator occurs in any position of a compound.

(1) Guanidine derivatives such as methyl guanidine (Japanese Patent Laid-Open Nos. 62-142114, JP-A 62-249908, JP-A-56614, JP-A-83059, JP-A 2-156, JP-A 2-765 Japanese Patent Application Laid-Open No. 2-42053, Japanese Patent Laid-Open No. 6-9380, Japanese Patent Laid-Open No. 5-505189).

(2) hydrazine derivatives such as sulfonylhydrazine.

(3) pyrazolone (Japanese Patent Laid-Open No. 6-287179), pyrazoline (Japanese Patent Laid-Open No. 10-167965), pyrazole (Japanese Patent Laid-Open No. 6-192089, Japanese Patent Laid-Open No. 6-298737, Japanese Patent Laid-Open No. 6-298738), Two nitrogen atoms such as imidazolidine (Japanese Patent Laid-Open Publication No. 5-201993, Japanese Patent Laid-Open Publication No. Hei 6-135968, Japanese Patent Laid-Open Publication No. Hei 7-133264, Japanese Patent Application Laid-Open No. 10-182460) and Hydantoin 5-membered heterocyclic compound having a compound.

(4) 5-membered heterocyclic compounds having three nitrogen atoms, such as triazole (Japanese Patent Application Laid-Open No. Hei 6-192089).

(5) Thiazoline (Japanese Patent Application Laid-Open No. Hei 10-167965), Thiazole (Japanese Patent Application Laid-Open No. Hei 4-9375, Japanese Patent Application Laid-Open No. Hei 9-59258), Japanese Patent Application Laid-Open No. Hei 5-201993; 5-membered heterocyclic compounds having one nitrogen atom and one sulfur atom, such as Japanese Patent Application Laid-Open No. Hei 7-133264 and 8-157473).

(6) 5-membered heterocyclic compounds having one nitrogen atom and one oxygen atom, such as oxazole (Japanese Patent Application Laid-Open No. Hei 9-59258).

(7) Nitrogen-containing six-membered heterocyclic compounds such as pyridine (Japanese Patent Laid-Open Publication No. Hei 10-158244, Japanese Patent Laid-Open Publication No. Hei 10-175954) and pyrimidine (Japanese Patent Laid-Open Publication No. Hei 7-500811).

(8) Nitrogen-containing condensed heterocyclic compounds such as indazole (Japanese Patent Laid-Open No. 6-287180), benzoimidazole (Japanese Patent Laid-Open No. 6-305964), and quinoline (Japanese Patent Laid-Open No. 3-161441).

(9) Sulfur-containing nitrogen-containing heterocyclic compounds such as benzothiazole (Japanese Patent Application Laid-Open No. Hei 6-305964).

(10) Sulfur-containing condensed heterocyclic compounds such as benzothiophene (JP-A-7-196498).

(11) Oxygen-condensed heterocyclic compounds, such as benzopyran (Japanese Patent Laid-Open No. 3-204874, Japanese Patent Laid-Open No. 4-308586).

(12) Nitrogen compounds, such as carbazoyl (Japanese Patent Laid-Open No. 2-156 and Japanese Patent Laid-Open No. 2-753), carbazin acid (Japanese Patent Laid-Open No. 2-167264), and hydrazine (Japanese Patent Laid-Open No. 3-148220).

(13) Quinones, such as benzoquinone (Japanese Patent Laid-Open No. 9-315960) and hydroquinone (Japanese Patent Laid-Open No. 5-9114).

(14) Aliphatic dicarboxylic acids (Japanese Patent Laid-Open No. Hei 1-56614, Japanese Patent Laid-Open No. 5-310565).

(15) Silicon-containing compound (Japanese Patent Application Laid-Open No. 62-249709).

(16) Organic germanium compounds (Japanese Patent Laid-Open No. 2-62885, Japanese Patent Laid-Open No. 5-255130, Japanese Patent Laid-Open No. 7-247296, Japanese Patent Laid-Open No. 8-59485).

(17) Flavonoids (Japanese Patent Application Laid-Open No. 3-240725, Japanese Patent Application Laid-Open No. 7-206838, Japanese Patent Application Laid-Open No. 9-241165, WO 94/04520).

(18) Alkylamines (Japanese Patent Application Laid-Open No. 6-206818, Japanese Patent Application Laid-Open No. 9-59233, Japanese Patent Application Laid-Open No. 9-40626, Japanese Patent Application Laid-Open No. 9-124471).

(19) Amino acids (Japanese Patent Laid-Open No. 4-502611, Japanese Patent Laid-Open No. 7-503713).

(20) Aromatic compounds, such as ascorroline (Japanese Patent Laid-Open No. Hei 6-305959), benzoic acid (WO 91/11997), and pyrrolonaphthyridinium (Japanese Patent Laid-Open No. Hei 10-158265).

(21) Polypeptides (JP-A-7-500580).

(22) Vitamins, such as pyridoxamine (WO97 / 09981).

(23) SH group-containing compounds such as glutathione, cysteine and N-acetylcysteine.

(24) SH group-containing proteins such as reduced albumin.

(25) Tetracycline compound (Japanese Patent Laid-Open No. 6-256280).

(26) Chitosans (Japanese Patent Laid-Open No. 9-221427).

(27) Tannins (Japanese Patent Application Laid-Open No. 9-40519).

(28) Quaternary ammonium ion containing compound.

(29) Biguanides, such as phenformin, buformin, or metformin.

(30) ion exchange resins.

(31) Inorganic compounds such as activated carbon, silica gel, alumina and calcium carbonate.

Such compounds include compounds generally known as Maillard inhibitors. The Maillard reaction is a non-enzymatic glycosylation reaction between reducing sugars such as glucose and amino acids or proteins. In 1912, Maillard reported the fact that browning occurs when the mixture of amino acids and reducing sugars is heated (Maillard, LC). , Compt.Bend.Soc.Biol., 72: 599 (1912), this Maillard reaction is involved in the browning changes, heating, storage, flavoring and protein denaturation of foods during heating and storage. Since doing so, research has been advanced in the field of food chemistry.

However, in 1968 glycosilhemoglobin (HbAlc), a small fraction of hemoglobin, was identified in vivo, and furthermore it was found to increase in diabetics (Rahbar, S., Clin. Chim. Acta, 22: 296 (1968). ), The significance of the Maillard response in vivo and the relationship between the onset of aging and the progression of aging such as diabetic complications and arteriosclerosis have been noted. Then, the search for a substance that inhibits the Maillard reaction in vivo was energetically performed, and the above-mentioned compounds were found as Maillard reaction inhibitors.

However, the fact that these Mailrad reaction inhibitors can remove the peritoneal dialysis fluid or blood-derived carbonyl compounds to improve the carbonyl stress state of peritoneal dialysis patients and inhibit the peritoneal dialysis complications caused by carbonyl stress , Was not known at all.

In addition, the carbonyl compound trapping agent of the present invention can be used in addition to the organic compounds represented by the aforementioned Maillard reaction inhibitors, polymer compounds such as ion exchange resins, or inorganic compounds such as activated carbon, silica gel, alumina and calcium carbonate. . These compounds are insoluble carbonyl compound trapping agents for the peritoneal dialysis solution, and the carbonyl compounds can be trapped using the carbonyl compound adsorption capacity. These are known as fillers for chromatography, but the fact that they are useful for improving the carbonyl stress state is not known.

Conventionally, adsorption-type blood purifiers using activated carbon have various endogenous and exogenous toxins and vascular function that increase in the early stages of blood poisoning during drug poisoning or hepatic coma and acute renal development in multiple organ failure. It is used as an adjuvant therapy of hemodialysis for the purpose of removing substances. However, it is not known at all that such an adsorption type blood purifier is effective for the removal of carbonyl compounds accumulated in the peritoneal cavity in peritoneal dialysis solution or during dialysis.

Further, Japanese Patent Application Laid-Open No. 58-169458 discloses a peritoneal dialysis solution containing a solid particulate water absorbing agent and an invention related to a peritoneal dialysis method using the peritoneal dialysis solution. However, the publication discloses that the solid particulate water absorbing agent is added for removal of creatinine and low molecular weight metabolites, and there is no description that this is useful for removing carbonyl compounds accumulated in the peritoneal cavity in peritoneal dialysis solution or during dialysis. Furthermore, neither the teaching nor the teachings of the improvement of the carbonyl stress state in a peritoneal dialysis patient by using the above-mentioned peritoneal dialysis method are provided.

The composition of the peritoneal dialysis solution serving as the base to which the carbonyl compound trapping agent in the present invention is added may be a known one. A general peritoneal dialysis solution is composed of a penetration regulator, a buffer, an inorganic salt, and the like. As the penetration regulator, sugars as listed above are used. As a buffer, the buffer system which gives the pH of the acidic side (pH 5.0-5.4) is mainly used in consideration of glucose stability. As a matter of course, when glucose is not added to the penetration regulator, a buffer that gives a pH of about 7.0, which is a more physiological pH, can be used. In addition, in order to use the pH of the neutral region while using glucose, a commercially available form of packaging for the buffer to adjust the pH at the time of use is also devised. In addition, in the present invention, since the carbonyl compound derived from the decomposition of glucose at the time of heat sterilization or long-term storage is removed, a buffer system giving a neutral pH that is difficult to formulate conventionally for the decomposition of glucose is preferable. Can be used. Furthermore, inorganic salts such as sodium chloride, calcium chloride, or magnesium chloride are usually added to the peritoneal dialysis solution. These salts are added in anticipation of improvement of biocompatibility by making peritoneal dialysis similar to physiological conditions.

The carbonyl compound trapping agent of the present invention can be added at the time of compounding the peritoneal dialysis solution, sealed as it is, and sterilized by heat for such a known formulation. By doing so, a preventive action can be expected for the production of carbonyl compounds from these main components that accompany the heat sterilization treatment. Further, the peritoneal dialysis solution may be accommodated in the divided chambers of the first chamber and the second chamber, the reducing sugar may be accommodated in the first chamber, the carbonyl compound trapping agent may be mixed in the second chamber, and mixed immediately before use. In this case, the carbonyl compound produced at the time of sterilization and storage is rapidly bonded with the mixed carbonyl compound trapping agent. After administration intraperitoneally, the excess carbonyl compound trapping agent captures the carbonyl compound derived from blood. The carbonyl compound trapping agent added to the peritoneal dialysis solution may be used alone or in combination of a plurality of kinds thereof.

On the other hand, various forms are considered as a contact method of an insoluble carbonyl compound trapping agent and a peritoneal dialysis solution to the carrier which fixed the carbonyl compound trapping agent, or a peritoneal dialysis solution. For example, a peritoneal dialysis solution is contained in a container in which a carbonyl compound trapping agent is immobilized therein, or a container containing a carbonyl compound trapping agent fixed in a carrier such as particles or fibers, and traps a carbonyl compound produced and accumulated during storage. You can. In the latter, the insoluble carrier can be separated from the peritoneal dialysis solution by filtration or the like. In addition, a bead, fibrous carrier or the like in which the carbonyl compound trapping agent is immobilized, or the insoluble carbonyl compound trapping agent is packed into a column for a carbonyl compound trap cartridge, and the peritoneal dialysis solution is brought into contact with the cartridge. You can also introduce. Such a cartridge is preferably filled with distilled water or the like beforehand in order to prevent air mixing at the start of peritoneal dialysis. When contacting the cartridge for carbonyl compound trap during intraperitoneal introduction, the carbonyl compound derived from the patient accumulated during dialysis cannot be removed, but the carbonyl compound in the dialysis solution can be removed. Alternatively, in the case of the peritoneal dialysis method in which the peritoneal dialysis solution is circulated in a closed system circuit using a small circulation pump, the carbonyl compound trap cartridge in which the carbonyl compound trapping agent is immobilized is provided in the circulation circuit, so that not only the peritoneal dialysis solution, Removal of carbonyl compounds that accumulate in the abdominal cavity during dialysis can also be achieved.

The carrier for immobilizing the carbonyl compound trapping agent in the present invention is not particularly limited as long as it is harmless to the human body and has a stability and stability as a material which is in direct contact with the peritoneal dialysis solution. For example, a synthetic or natural organic high molecular compound And inorganic materials such as glass beads, silica gel, alumina, activated carbon, and the like coated with polysaccharides, synthetic polymers, and the like on the surfaces thereof. The insoluble carbonyl compound trapping agent is a substance permeability, chemical compatibility, protective strength, adsorption capacity, or carbon for a carrier for immobilizing the carbonyl compound trapping agent or a self-peritoneal dialysis solution. It can increase the specificity for the carbonyl compound.

Examples of the carrier made of a high molecular compound include polymethyl methacrylate polymers, polyacrylonitrile polymers, polysulfone polymers, vinyl polymers, polyolefin polymers, fluorine polymers, polyester polymers and polyamides. Polymers, polyimide polymers, polyurethane polymers, polyacrylic polymers, polystyrene polymers, polyketone polymers, silicone polymers, cellulose polymers, chitosan polymers, and the like. Specifically, polysaccharides and derivatives thereof such as agarose, cellulose, chitin, chitosan, cephalos, and dextran, polyester, polyvinyl chloride, polystyrene, polysulfone, polyethersulfone, polypropylene, polyvinyl Alcohol, polyallyl ether sulfone, polyacrylic acid ester, polymethacrylic acid ester, polycarbonate, acetylated cellulose, polyacrylonitrile, polyethylene terephthalate, polyamide, silicone resin, fluorine resin, polyurethane, polyetherurethane, poly Acrylamide, derivatives thereof, etc. are mentioned. These polymeric materials can be used individually or in combination of 2 or more types. In the case of combining two or more kinds, the carbonyl compound trapping agent is immobilized on at least one of them. The immobilized carbonyl compound trapping agent may be immobilized alone, or two or more kinds thereof may be immobilized. In addition, the said polymeric material is used as a homopolymer, and can also be set as a copolymer with a heteropolymer. In addition, an appropriate improving agent may be added or a modification treatment such as radiation crosslinking or peroxide crosslinking may be performed.

There is no restriction | limiting in the shape of a support | carrier, For example, a film form, a fibrous form, a hollow fiber form, a nonwoven fabric form, a porous form, honeycomb formation, etc. are mentioned. These carriers can control the contact area with the peritoneal dialysis fluid by varying the thickness, surface area, thickness, length, shape and / or size. In addition, the carbonyl compound trapping agent may be fixed to the inner wall of the container containing the peritoneal dialysis fluid or the inside of the peritoneal dialysis fluid circulation circuit.

When immobilizing the carbonyl compound trapping agent on the carrier, a known method such as physical adsorption, biochemical specific bonding, ionic bonding, covalent bonding, grafting or the like may be used. If necessary, a spacer may be introduced between the carrier and the carbonyl compound trapping agent. When elution from the carrier becomes a problem, such as when the carbonyl compound trapping agent is toxic, it is preferable that the carbonyl compound trapping agent is covalently immobilized to the carrier in order to reduce the elution amount as much as possible. When the carbonyl compound trapping agent is covalently bonded to the carrier, a functional group present in the carrier may be used. Examples of the functional group include a hydroxyl group, an amino group, an aldehyde group, a carboxyl group, a thiol group, a hydroxyl group, a silanol group, an amide group, an epoxy group, a succinylimide group, and the like, but are not limited thereto. Examples of covalent bonds include ester bonds, ether bonds, amino bonds, amide bonds, sulfide bonds, imino bonds, and disulfide bonds.

As the carrier on which the carbonyl compound trapping agent is immobilized, commercially available ones such as polystyrene carriers having a sulfonylhydrazine group (PS-TsNHNH2, ARGONAUT TECHNOLOGIES) can be used.

As for sterilization of the carrier on which the carbonyl compound trapping agent is immobilized based on the present invention, an appropriate sterilization method is selected from known sterilization methods in accordance with the kind of carbonyl compound trapping agent or carrier. Sterilization includes high pressure steam sterilization, gamma irradiation sterilization and gas sterilization. A cartridge filled with an insoluble carbonyl compound trapping agent or a carbonyl compound trapping agent bound to a carrier may be prepared, and both may be sterilized simultaneously while being connected to a container containing peritoneal dialysis solution.

When there is little carbonyl compound trapping agent in contact with a peritoneal dialysis liquid, the carbonyl compound in a peritoneal dialysis liquid cannot be removed sufficiently. In general, it is difficult to predict the amount of carbonyl compound in the peritoneal dialysis in advance, so it is effective to maintain the activity of the large amount of carbonyl compound trapping agent as long as the safety of the patient can be guaranteed. . The capacity of the carbonyl compound trapping agent can be adjusted by changing the amount of immobilization of the carbonyl compound trapping agent to the carrier or the amount of the carrier on which the carbonyl compound trapping agent is immobilized.

Alternatively, the carbonyl compound trapping agent may be injected into the peritoneal dialysis circuit equipped with a suitable mixing connector. In this case, the carbonyl compound produced at the time of sterilization and storage is captured in a circuit.

In addition, the carbonyl compound trapping agent may be directly administered intraperitoneally and mixed with the peritoneal dialysis solution intraperitoneally. At this time, the carbonyl compound derived from peritoneal dialysis solution and blood is inactivated intraperitoneally.

In addition, by administering the carbonyl compound trap agent into the peritoneal dialysis patient by intravenous injection or the like before injecting the peritoneal dialysis solution into the patient or intraperitoneal retention, the carbonyl stress state in the abdominal cavity can be improved.

Below, the preferable embodiment of the peritoneal dialysis liquid which concerns on this invention is shown concretely.

Generally, the following prescription is used for the composition of the dialysis liquid used as a base.

Their prescription is general, and in reality, a more appropriate composition is employed according to the symptoms of the patient.

Glucose 1-5% w / m

Sodium ion 100-150meq

Potassium Ion 0-0.05meq

Magnesium ion 0-2meq

Calcium ion 0-4meq

Chlorine Ion 80-150meq

Buffer 10-50mM

(Organic acids such as lactic acid, citric acid, nitric acid, acetic acid, pyrvic acid and succinic acid)

To the basic prescription as described above, an effective amount of the carbonyl compound trapping agent according to the present invention is added. For example, when aminoguanidine is added, it is added so as to have a concentration of 1 mM or more, preferably 10 mM or more, more preferably 10 mM or more and 100 mM or less. When the addition amount is small, the carbonyl compound trapping agent is consumed by the carbonyl compound generated during manufacture and storage, and the carbonyl compound leaching from the patient's blood or tissue into the dialysate may not be treated during actual dialysis. It is expected. In particular, it is difficult to predict the amount of carbonyl compound leaching from the blood or tissue of the patient in advance, so that it is possible to maintain the activity of a large amount of carbonyl compound trapping agent as long as possible to ensure safety for the patient. effective. It is known that aminoguanidine is lowly toxic to animals. According to the "Registry of Toxic Effect of Chemical Substances" (1978), the half dose of aminoguanidine is 1258 mg / in rat subcutaneous injection. kg and 963 mg / kg in mice. Moreover, it is excellent in water solubility. In addition, in the case of OPB-9195, it is added so that it may become 1mM or more, Preferably it is 10mM or more, More preferably, it is 10mM or more and 100mM or less.

The peritoneal dialysis solution according to the present invention formulated as described above is filled into a suitable sealed container and sterilized. Heat sterilization such as high pressure steam sterilization or hot water sterilization is effective for sterilization treatment. In this case, a container having a strength that withstands transportation even after sterilization is used without elution of harmful substances at high temperatures. Specific examples thereof include a flexible plastic bag made of polyvinyl chloride, polypropylene, polyethylene, polyester, ethylene vinyl acetate copolymer, and the like. In addition, in order to avoid deterioration of the liquid due to the influence of outside air, the container filled with the peritoneal dialysis solution may be further packed with a packaging material having high gas barrier property.

Instead of heat sterilization, filter sterilization may be performed. For example, sterilization is performed by filtration using a precision filter equipped with a membrane filter having a pore size of about 0.2 μm. In this case, generation | occurrence | production of the carbonyl compound resulting from heating can be prevented. The peritoneal dialysis fluid that has been sterilized is filled in a container such as a flexible plastic bag and then sealed. Since the series of treatments from sterilization to transportation are no different from the production of the current dialysis solution, the peritoneal dialysis solution according to the present invention can be produced by the same process.

In the case of sterilization by heat sterilization including high pressure heat sterilization, if the carbonyl compound trapping agent used is sufficiently stable for treatment such as heating, the carbonyl compound trapping agent is added in advance at the time of compounding the peritoneal dialysis solution, Heat sterilization can be performed. In this way, generation and accumulation of the carbonyl compound derived from the dialysate at the time of heating can be suppressed. Of course, the carbonyl compound trapping agent also functions during storage or in a peritoneal dialysis solution to suppress the production and accumulation of carbonyl compounds.

If the carbonyl compound trapping agent used is unstable in heat sterilization, a sterilization method that does not require heating may be used. Such sterilization includes, for example, filter sterilization. In addition, the carbonyl compound trapping agent may be added later to the peritoneal dialysis solution previously sterilized by heat. The time of addition is not specifically limited. For example, it is preferable to add the carbonyl compound trapping agent after sterilizing the liquid, since the production and / or accumulation of the carbonyl compound can be suppressed not only during peritoneal dialysis but also during peritoneal dialysis solution storage before dialysis.

Alternatively, the carbonyl compound trapping agent may be added immediately before or simultaneously with peritoneal dialysis. For example, the base solution and the carbonyl compound trapping agent are separately contained in the above-described soft plastic bag or the like until just before use, and both are aseptically mixed immediately before the start of peritoneal dialysis. For this purpose, a flexible plastic bag divided by a peelable adhesive portion as disclosed in Japanese Patent Application Laid-Open No. 63-19149 is preferably used.

In the middle of the peritoneal dialysis circuit, a connector member for mixing may be provided to inject a carbonyl compound trapping agent from the connector.

Such a method for preparing the peritoneal dialysis solution can be used not only for the heat unstable carbonyl compound trapping agent but also for the heat stable carbonyl compound trapping agent.

Furthermore, in the case of the peritoneal dialysis method in which the peritoneal dialysis solution is circulated in a closed system circuit using a small circulation pump, a filter in which a carbonyl compound trapping agent is filled in any one of the circuits may be provided.

The peritoneal dialysis solution of the present invention is used for the same peritoneal dialysis treatment as the current peritoneal dialysis solution. That is, a proper amount of the peritoneal dialysis solution according to the present invention is injected into the peritoneal cavity of the dialysis patient, and the low molecular weight component in vivo is transferred into the peritoneal dialysis fluid through the peritoneum. Peritoneal dialysis fluid circulates intermittently and continues dialysis according to the patient's condition. At this time, the carbonyl compound, together with a dialysis component such as creatinine, inorganic salts, or chlorine ions, transfers into the peritoneal dialysis solution in the blood or in the peritoneum. At the same time, by the action of the carbonyl compound trapping agent, the harmful activity to the living body is lost and innocuous.

Best Mode for Carrying Out the Invention

Hereinafter, although an Example demonstrates this invention concretely, this invention is not restrict | limited by these Examples.

Example 1 Measurement of Carbonyl Compounds in Peritoneal Dialysis Solution and Peritoneal Dialysis Drainage

In order to prove the occurrence of carbonyl stress in the abdominal cavity, the amount of carbonyl compound in the peritoneal dialysis drainage was measured according to the following experimental method.

1) Measurement of Carbonyl Compound

Peritoneal dialysis solution (Baxter Ltd .; Dianeal PD-2 2.5) and peritoneal dialysis patients were treated with the above-mentioned peritoneal dialysis solution to obtain 400 μL of peritoneal dialysis drainage after overnight storage in the peritoneal cavity, and 1.5mM 2,4-dinitrophenylhydrazine ( 2,4-DNPH) (manufactured by Wako Pure Chemical Industries, Ltd.) was mixed with 400 µL of 0.5 N hydrochloric acid solution, stirred at room temperature for 30 minutes, and the carbonyl compound and 2,4-DNPH in the liquid were reacted. Next, 40 µL of 1 M acetone aqueous solution was added, and stirred at room temperature for 5 minutes to remove excess 2,4-DNPH with acetone, followed by washing three times with 400 µL of n-hexane, followed by 360 nm water layer. Absorbance was measured with a spectrophotometer for microplates (Japan Molecular Devices co .; SPECTRAmax250).

2) Preparation of calibration curve

Glucose aqueous solutions of various concentrations were prepared and operated in the same manner as in 1) to prepare a calibration curve for the amount of glucose and glucose-derived carbonyl compounds.

3) Determination of Carbonyl Compounds

Glucose concentrations in the peritoneal dialysis solution and the peritoneal dialysis drainage were measured using a glucose measurement kit (glucose CI-Test Wako). The quantity of the carbonyl compound derived from glucose was calculated | required from the analytical curve. The amount of carbonyl compound derived from glucose was subtracted from the amount of total carbonyl compound in the liquid to give an amount of carbonyl compound.

The obtained result is shown in FIG. In the peritoneal dialysis fluid after overnight storage in the abdominal cavity, the amount of carbonyl compound was about five times as compared with the peritoneal dialysis fluid before administration, and the transition of the carbonyl compound from the blood into the abdominal cavity was shown.

Example 2 Histological Localization of Carbonyl Modified Protein in Peritoneum of Peritoneal Dialysis Solution Patients

Carbonyl compounds in the peritoneal tissues of peritoneal dialysis patients were examined by immunostaining with malondialdehyde as an index.

For peritoneal dialysis of peritoneal dialysis patients (50-year-old male, peritoneal dialysis 5 years) to Horie et al. (Horie, K. et al., J. Clin. Invest., 100, 2995-3004 (1997)) Therefore, immunostaining was performed using mouse antimalondialdehyde monoclonal antibody as the primary antibody. As a result, strong positive findings were observed on the detached connective tissue under the mesothelial cells and the thickened blood vessel wall (FIG. 2).

Malondialdehyde (MDA) is a carbonyl compound produced by the decomposition of lipid peroxide. Therefore, similarly immunostaining was carried out using 4-hydroxy-2-nonenal, a lipid peroxidation product other than MDA, and an antibody against carboxymethyl lyzine or pentosidine produced as a result of glycosylation reaction. The same sites as the positive sites indicated by were positive. From this fact, it was found that the peritoneal tissue of the peritoneal dialysis patient was subjected to protein modification due to the promotion of carbonyl stress due to the carbonyl compound derived from the lipid peroxidation product and the glycosylation product.

Example 3 Peritoneal Cell Injury by Methylglyoxal

The decrease in the ultrafiltration function of the peritoneum by prolonged peritoneal dialysis supports the increase of peritoneal surface area that allows mass exchange by diffusion (Krediet, RT, 1999, Kidney Int. 55: 341-356; Heimburger, O. et. al., 1990, Kidney Int 38: 495-506; Imholz, AL et al., 1993, Kidney Int. 43: 1339-1346; Ho-dac-Pannekeet, MM et al., 1997, Perit.Dial.Int. 17: 144-150). That is, the glucose in the peritoneal dialysis solution diffuses and flows out of the intraperitoneal cavity, thereby degrading the dialysis function due to the infiltration pressure gradient. This can also be explained by the increase in blood vessel surface area in the peritoneum. In this condition, vascular endothelial growth factor (VEGF) is considered to play an important role. VEGF promotes vascular permeability (Senger, DR et al., 1983, Science 219: 983-985; Connolly, DT et al., 1989, J. Biol. Chem. 264: 20017-20024), and synthesis of NO Stimulation of blood vessels (Hood, JD et al., 1998, Am. J. Physiol. 274: H1054-1058) to induce an inflammatory response (Clauss, M. et al., 1990, J. Exp. Med. 172: 1535-1545; Melder, RJ et al., 1996, Nat. Med. 2: 992-997). In addition, VEGF is a potent angiogenic factor and has a function of repairing vascular injury (Thomas, KA, 1996, J. Biol. Chem. 271: 603-606; Ferrara, N. et al., 1997, Endocr, Rev 18: 4-25; Shoji, M. et al., 1998, Am. J. Pathol, 152: 399-411). Therefore, the effects of glucose degradation products contained in the dialysate on VEGF production were verified.

<3-1> Expression of VEGF in Peritoneal Epithelial and Vascular Endothelial Cell Culture in the Presence of Glucose Degradation Products

Peritoneum was taken from male CD (SD) IGS rats (Charles-River, Kanagawa, Japan) at 6 weeks old. Mesothelial cells were isolated based on Hjelle et al. (Hjelle. JT et al., 1989, Perit.Dial. Int. 9: 341-347), and Dalbecco's modified medium containing 10% fetal bovine serum. Incubated in Eagle's medium (DMEM). Mesothelial cells of the seventh to tenth passages were prepared by glucose degradation products of various concentrations (glyoxal, methylglyoxal (Sigma, St. Louis, MO), or 3-deoxyglucoson (Fuji Memorial Research Institute, Otsuka Pharmaceutical Co. (Provided by Kyoto, Japan)), and incubated for 3 hours in a CO ₂ incubator. Expression analysis of VEGF mRNA was performed by semiquantitative RT-PCR. Whole RNA was isolated from mesothelial cells using the Rneasy Mini Kit (Qiagen, Germany). 5 μg of RNA was reverse transcribed using _12-18 primers (Gibco BRL, Gaithersburg, MD) and 200 units of RNase H-free reverse transcriptase (Superscript II: Gibco BRL), and PCR amplification was described above. Conditional (Miyata, T. et al., 1994, J. Clin. Invest. 93: 521-528). The oligonucleotide primer sequence used was amplified by 310 bp in 5'-ACTGGACCCTGGCTTTACTGC-3 '(SEQ ID NO: 1) and 5'-TTGGTGAGGTTTGATCCGCATG-3' (SEQ ID NO: 2) for the primer used for amplification of rat VEGF. Got it. Primers used for amplification of rat glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were 5'-CCTGCACCACCAACTGCTTAGCCC-3 '(SEQ ID NO: 3) and 5'-GATGTCATCATATTTGGCAGGTT-3' (SEQ ID NO: 4) 322 bp fragment was amplified. G3PDH was used as an internal standard for RNA, and RNA levels were compared between different samples. Each sample was first determined a suitable number of cycles using a DNA thermal cycler (Perkin Elmer Ceus, Norwork, CT) at the conditions of 94 ° C 0.5 minutes, 64 ° C 1 minute, 72 ° C 1.5 minutes. As a preliminary experiment, reverse transcription and PCR amplification were performed under conditions of 16, 18, 21, 25, 28, 31 and 34 cycles using various RNA amounts. As a result, a signal of a PCR product quantitatively correlated with the added RNA was obtained under conditions of 30 cycles for VEGF mRNA amplification and 21 cycles for amplification of G3PDH mRNA. PCR products were electrophoresed with 1.5% agarose gel, stained with ethidium bromide, and then quantified by measuring signal intensity using a quantitative program (NIH image). The experiment was performed at each glucose decomposition product concentration. MRNA was quantified in three consecutive experiments and averaged for each experiment. A total of 3 to 4 independent experiments were conducted under each experimental condition. Results were averaged and expressed as mean ± SD. Statistical significance was assessed by ANOVA. When significant differences were found by this analysis, the results obtained at different methylglyoxal concentrations were compared by Scheffe's t-test.

At various concentrations from 0 to 400 μM, glyoxal or 3-deoxyglucosone did not alter the expression of VEGF (FIGS. 3A and B). Only methylglyoxal stimulated the expression of VEGF mRNA at a concentration of 400 μM (P <0.0005) (FIG. 3C). No PCR product was generated from the RNA sample that was not reverse transcribed. All cells were viable. When the mesothelial cells were cultured in the presence of high concentration of 3-deoxyglucosone (0.625, 2.5 and 5 mM), there was a decrease in the viability of the cells (cell survival rate was 0.625, 2.5, 5 mM in the presence of 3-deoxyglucosone, 80,55 and 8%, respectively). Because cell viability decreased, VEGF mRNA expression could not be measured. From these observations, only methylglyoxal was used for the following experiments.

The release of VEGF protein into the culture supernatant of human microvascular endothelial cells cultured for 24 hours in the presence of various concentrations of methylglyoxal was measured by ELISA. Human microvascular endothelial cells were purchased from Kurabo (Osaka, Japan) and cultured in VEGF deleted EGM-2 medium (Takara, Tokyo, Japan). Incubation with methylglyoxal was performed in the same manner as the rat peritoneal mesothelial cells. The VEGF protein was quantified by the enzyme-linked immunosorbant assay (ELISA) according to the attached instructions using the kit (Quantikine: R & D Systems, Minneapolis, USA) for the culture supernatant prepared twice in succession. . The experiment was repeated three times, and the results were statistically analyzed in the same manner as above.

As a result, an increase in VEGF was recognized in dose-dependent manner by the addition of methylglyoxal to the medium (Fig. 4). In the absence of methylglyoxal, no release of VEGF was detected. That is, even at the protein level, the dose-dependent VEGF production and release was stimulated by methylglyoxal.

Next, expression of VEGF mRNA in endothelial cells cultured in the presence of methylglyoxal at various concentrations (0-400 µM) was examined. The experiment was conducted in the same manner as in the case of rat mesothelial cells. However, the fragments amplified using primers of 5'-GGCAGAATCATCACGAAGTGGTG-3 '(SEQ ID NO: 5) and 5'-CTGTAGGAAGCTCATCTCTCC-3' (SEQ ID NO: 6) were 271 bp for amplification of human VEGF. The same primers as rats were used for amplification of human G3PDH. As a result of the analysis, the expression of VEGF mRNA was found to increase dose-dependently (Fig. 5).

<3-2> Expression of VEGF in Peritoneal Tissues of Rats Intraperitoneally Administered with Methylglyoxal

In order to further investigate in vivo the biological effect of methylglyoxal on the expression of VEGF mRNA in the peritoneum, an experiment was conducted in which rats were infused with various amounts of methylglyoxal for 10 days. Six-week-old male CD (SD) IGS rats received 50 ml / kg of saline containing various concentrations of methylglyoxal intraperitoneally for 10 days. The peritoneum was isolated from the abdominal wall to examine the expression of VEGF mRNA. The experiment was performed in the same manner as the in vitro experiment of the rat mesothelial cells. However, ISOGEN (Nippon Gene, Tokyo, Japan) was used to extract mRNA from peritoneal tissue. PCR amplification was performed under conditions of 28 cycles for VEGF mRNA amplification and 16 cycles for amplification of G3PDH mRNA.

As shown in Fig. 6, the methylglyoxal concentration significantly increased the expression of VEGF mRNA in the peritoneal peritoneal sample (P <0.05). Under light microscopy, no changes were found in the peritoneal tissue (vessels, blood vessel walls, gaps and mesothelial cells were normal).

<3-3> Immunostaining of VEGF and Carboxymethyl Lysine (CML) in Peritoneal Tissue in Patients with Long-term Peritoneal Dialysis

The distribution of VEGF and carboxymethyl lysine in the peritoneal tissues of nine patients with peritoneal dialysis was examined by immunohistochemical analysis. Carboxymethyl lysine is derived from glucose degradation products such as glyoxal and 3-deoxyglucoson (Miyata, T. et al., 1999, Kidney Int. 55: 389-399). Thus, anticarboxymethyl lysine antibodies are used as markers for proteins modified with glucose breakdown products.

After informed consent was obtained, peritoneal tissue was isolated from nine non-diabetic peritoneal dialysis patients at catheter reinsertion (Table 1). The need for catheter reinsertion is caused by damage to the catheter, misalignment and / or occlusion. There were no patients with peritonitis. Two normal men (48 and 58 years old) with normal renal function were isolated from normal peritoneal tissue at the time of abdominal incision.

A 2 μm thick piece of peritoneal tissue was mounted on a slide coated with 3-aminopropyltriethoxysilane (Signa), deparaffinized, and then passed through distilled water, followed by Pronase (0.5 mg / μl: Dako, Glostrup, Denmark). ) Was incubated for 15 minutes at room temperature with a buffer solution (0.05M Tris-HCl (pH 7.2), 0.1M NaCl). Slides were washed with PBS containing 0.5% Tween20 and blocked for 2 hours with 4% skim milk, followed by anti-VEGF rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-AGE mouse IgG ( Ikeda, K. et al., 1996, Biochemistry 35: 8075-8083) (carboxymethylazine is epitope: Miyata, T. et al., 1997, Kidney Int. 51: 1170-1181) Incubated overnight at 4 ° C. in a humid chamber. After washing the pieces, incubate at 1: 100 dilution with peroxidase-linked goat anti-rabbit IgG or peroxidase-linked goat anti-mouse IgG (Dako) for 2 hours at room temperature, containing 3,3 'containing 0.003% H ₂ O ₂ . The signal was detected with diaminobenzidine solution. In addition, histological analysis was performed by peroxo-acid staining. Immunostaining was observed independently in two patients, and signal intensity and distribution were evaluated.

The results are shown in Table 1. In the table, "normal 1" and "normal 2" indicate samples derived from a subject with normal renal function, and "PD1" to "PD9" indicate samples derived from peritoneal dialysis patients. In the peritoneal tissue images (PD6 in Table 1) of representative long-term peritoneal dialysis patients, interstitial fibrosis, vascular wall thickness, and hyalinosis were recognized. Mesothelial cells and blood vessel walls were co-localized with VEGF and carboxymethyl lysine. In the mesothelial layer, the signal of VEGF was weaker than that of carboxymethyl lysine. The results were the same for the other eight patients. In normal peritoneal samples (normal 2 in Table 1), VEGF was present only in the vessel wall, compared to peritoneal dialysis samples, and was not detected in the mesothelial layer. Carboxymethyl lysine was not detected in the mesothelial layer, and the signal of the blood vessel wall was extremely weak. Their observations were the same for the other control normal samples. When normal mouse IgG was used, no immunostaining was detected. Thus, the fact that the expression of VEGF in the mesothelioma and coadministration of carboxymethyl lysine is seen only in the peritoneal dialysis patients. Thus, peritoneal dialysis results in the degradation of glucose degradation products in the peritoneal dialysis solution. This suggests that VEGF production is actually enhanced.

Immunohistochemical Detection of CML and VEGF in Peritoneal Tissue of Peritoneal Dialysis Patients Sample Gender Age Peritoneal Dialysis Period CML VEGF (Month) Mesothelial Vessel Wall Mesothelial Vessel Wall Normal 1 M 48--±-+ Normal 2 M 58--±-+ PD1 F 53 3 + + + + PD2 M 44 4 + + + + PD3 M 43 45 + + + + PD4 F 54 60 ++ ++ ++ + PD5 M 52 70 ++ ++ + + PD6 M 51 90 ++ ++ ++ ++ PD7 M 45 105 ++ ++ ++ PD8 M 62 108 ++ ++ ++ PD9 M 66 110 ++ ++ ++ ++

-: Negative, ±: extremely weak signal, +: positive, ++: strongly positive

The above results support for the first time that peritoneal dialysis patients are placed under carbonyl stress by glucose degradation products in peritoneal dialysis solution, and also demonstrate for the first time that methylglyoxal enhances the production of VEGF into peritoneal cells. It is suggested that at least a part of the cause of the degradation of glucose permeate contained in the peritoneal dialysis solution is due to the enhancement of VEGF production and the accompanying stimulation of angiogenesis.

Example 4 Addition Effect of Carbonyl Compound Trap Agent on Dialysis Fluid Drainage in Peritoneal Dialysis Patients

<4-1>

Pentosidine is one of the AGE constructs, and about 20 times the accumulation in the blood of renal failure patients is comparable to that of the average healthy body (Miyata, T. et al., J. Am. Soc. Nephrol., 7: 1198-1206 (1996). When the drainage of peritoneal dialysis patients was incubated at 37 ° C, the effect of the addition of aminoguanidine to the pentosidine and the carbonyl group (protein carbonyl) present on the protein was examined.

After centrifugation of the peritoneal dialysis patient overnight, the supernatant was filtered and sterilized (pore 0.45 µm), and aminoguanidine (curing agent) was added to a concentration of 0, 1, 10, 100 mM, and incubated at 37 ° C. . The incubation period was 1 to 2 weeks when pentosidine was measured and 2 weeks when protein carbonyl was measured. The measurement of pentosidine was quantified by HPLC (Shimazu Corporation, LC-10A) after hydrolysis of the protein at 110 ° C. in 6N HCl (T.Miyata et al., 1996, J. Am. Soc. Nephrol, 7). : 1198-1206, T. Miyata et al., 1996, Proc. Natl. Acad. Aci. USA, 93: 2353-2358). The protein carbonyl was measured by reacting with 2,4-dinitrophenylhydrazine (2,4-DNPH) (made by Wakojunyaku) and then measuring the absorbance of the hydrazone produced by reacting with a carbonyl group (Japan Molecular Devices co. (SPECTRAmax 250) (Levine, RL et al., Methods Enzymol., 233, 346-357 (1994)).

As a result, the inhibitory effect of aminoguanidine was observed in a concentration-dependent manner for the production of pentosidine (FIG. 8). Similarly, the inhibitory effect of aminoguanidine was recognized in a concentration-dependent manner in the production of protein carbonyl (Fig. 9).

<4-2>

Next, the effect of the addition of aminoguanidine to the amount of carbonyl compounds other than glucose in the overnight storage drainage of peritoneal dialysis patients was examined according to the following experimental method.

(A) Incubation of Peritoneal Dialysis Drainage

The drainage of the peritoneal dialysis patient was collected, filtered through a filter with a pore size of 0.45 µm, and aminoguanidine (copper curing agent) was added so as to have concentrations of 0, 10, 50, and 250 mM, to prepare a sample solution. 1 ml of the sample solution was taken, dispensed into a screw tube with a screw cap, and then incubated at 37 ° C for 15 hours. In addition, the sample solution was stored at -30 degreeC until just before incubation.

(B) Determination of Carbonyl Compounds

1) Measurement of sample solution

Take 400 μL of sample solution, mix 1.5 mM 2,4-DNPH (made by Wako Pure Chemical) with 400 μL of 0.5 N hydrochloric acid solution, stir at room temperature for 30 minutes, and mix the carbonyl compound and 2,4-DNPH in the sample solution. Reacted. Next, 40 µL of an aqueous 1 M acetone solution was added and stirred at room temperature for 5 minutes to remove excess 2,4-DNPH with acetone, and then the sample solution was washed three times with 400 µL of n-hexane. Absorbance at 360 nm was measured with a spectrophotometer for microplates (Japan Molecular Devices co .; SPECTRAmax250).

2) Preparation of calibration curve

Glucose aqueous solutions of various concentrations were prepared and operated in the same manner as in 1) to prepare a calibration curve for the amount of glucose and glucose-derived carbonyl compounds.

3) Determination of Carbonyl Compounds

Glucose concentration in the sample solution was measured using a glucose measurement kit (wako Pure Chemical, Glucose CII-Test Wako). The amount of carbonyl compounds derived from glucose was calculated | required from the analytical curve. The amount of carbonyl compound derived from glucose was subtracted from the amount of total carbonyl compound in the peritoneal dialysis drainage to obtain the amount of carbonyl compound.

The results are shown in FIG. The amount of carbonyl compound other than glucose in the peritoneal dialysis drainage of peritoneal dialysis patients decreased as the concentration of aminoguanidine was increased or decreased incubated at 37 ° C for 15 hours.

From these results, it became clear that the addition of the carbonyl compound trapping agent to the peritoneal dialysis solution or administration to the patient was effective for suppressing the production and / or accumulation of the carbonyl compound in the peritoneal dialysis solution injected intraperitoneally. As a result, the carbonyl compounds derived from the peritoneal dialysis fluid and the blood derived from the peritoneum are removed, and the carbonyl stress state of the peritoneal dialysis patient is improved.

Example 5 Addition Effect of Carbonyl Compound Trap Agent in the Heat Sterilization Process of Peritoneal Dialysis Solution

Peritoneal dialysis solution contains high concentration (1.35 ~ 4.0w / v%) glucose as penetration control agent. Glucose is unstable to heat and degrades during heat sterilization or storage. As a decomposition product of glucose, 5-hydroxymethylfurfural (5HMF), levulic acid, acetaldehyde and the like have been reported to be produced (Richard, JU et al., Fund.Appl. Toxic., 4: 843-853 (l984), Nilsson, CB et al., Perit.Dial.Int., 13: 208-213 (1993). The effect of inhibiting the production of the amino guanidine carbonyl compound during the heat sterilization of the peritoneal dialysis solution was examined by measuring the amount of 5-HMF and carbonyl compound. In addition, since the degree of decomposition of glucose was affected by the liquid pH, two types of peritoneal dialysis fluids with acidic pH (pH 5.3) and neutral pH (pH 7.0) before sterilization were prepared and used for the experiment. Sterilization temperature was 121 degreeC.

<5-1>

5-HMF was measured using high performance liquid chromatography (manufactured by Shimadzu Corporation, LC-10A) (Nilsson, CB et al., Perit.Dial. Int., 13: 2O8-213 (1993)). .

As a result, both the acidic environment (Fig. 11) and the neutral environment (Fig. 12), the inhibitory effect of aminoguanidine was observed in a concentration-dependent manner on the production of 5-HMF.

<5-2>

Quantitative determination of the carbonyl compound in the peritoneal dialysis solution was performed by reacting with 2,4-DNPH and measuring the absorbance as in Example 1 (Levine, RL et al., Methods Enzymol., 233: 346-357). (l994)). Sterilization temperature was 121 degreeC.

As a result, also regarding the amount of carbonyl compounds, the inhibitory effect of aminoguanidine was recognized in a concentration-dependent manner (Fig. 13).

From these results, it is evident that the addition of a drug that inhibits the production of the carbonyl compound in the peritoneal dialysis solution is very effective because it suppresses the production and / or accumulation of the carbonyl compound in the peritoneal dialysis solution.

Example 6 Addition Effect of Carbonyl Compound Trap Bead on Pentosidine Formation in Peritoneal Dialysis Solution

The removal effect of the carbonyl compound in the peritoneal dialysis solution was examined using a combination of a sulfonylhydrazine group and a crosslinked polystyrene resin (PS-TsNHNH2, ARGONAUT TECHNOLOGlES) as a carbonyl compound trap bead. The peritoneal dialysis solution containing the peritoneal dialysis solution and the carbonyl compound trap bead was incubated at 37 ° C to confirm the inhibitory effect of pentosidine formation. 100 μl of dimethyl sulfoxide was added to the tube containing the carbonyl compound trap bead and swelled, followed by 800 μL of peritoneal dialysis solution (Baxter Ltd .; Dianeal PD-4, 1.5) and 200 μL of bovine serum albumin at 150 mg / mL concentration. In addition, it was incubated for one week at 37 degreeC. After incubation, beads were removed using a centrifugal filter tube (Millipore co .; UFC30W00) with a pore size of 0.22 μm. Next, 50 µl of 10% trichloroacetic acid was added to 50 µl of the beads from which the beads were removed, and the protein was precipitated by centrifugation. The protein was washed with 300 µl of 5% trichloroacetic acid and dried. Next, 100 µl of 6N HCl was added and heated to 110 ° C for 16 hours, and then pentosidine was quantified by HPLC (T. Miyata et al., 1996, J. Am. Soc. Nephrol., 7: 1198-1206). , T, Miyata et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 2353-2358).

The amount of pentosidine produced when incubated at 37 degreeC is shown in FIG. It was found that the addition of carbonyl compound trap beads significantly inhibited the production of pentosidine.

Example 7 Effects of Carbonyl Compound Trap Beads on the Amount of Carbonyl Compounds in Peritoneal Dialysis Solution

The removal effect of the carbonyl compound in the peritoneal dialysis solution by the carbonyl compound trap bead was examined. To the tube containing the carbonyl compound trap bead (PS-TsNHNH2, ARGONAUT TECHNOLOGIES), swelled by adding 100 µl of dimethyl sulfoxide, and 900 µL of peritoneal dialysis solution (Baxter Ltd .; Dianeal PD-4, 1.5) was added to the rotator. It stirred at room temperature for 16 hours using the rotator. Next, the suspension containing the carbonyl compound trap beads was filtered through a centrifugal filter tube (Millipore co; UFC30GV00) having a pore size of 0.22 µm, and the amount of carbonyl compound in the filtrate was measured as follows.

<Quantification of Carbonyl Compounds>

1) Measurement of sample solution

After mixing 200 µL of the sample solution and 200 µL of a solution (0.025%) in 2,4-DNPH dissolved in 0.5N hydrochloric acid, the mixture was reacted at 30 ° C for 30 minutes. Next, 20 µL of an aqueous 1 M acetone solution was added and incubated at 30 ° C. for 10 minutes, followed by washing three times with 200 µL of n-hexane. Furthermore, 200 µL of octanol was added to the aqueous layer to extract hydrazone, and the absorbance at 360 nm was measured with the microplate spectrophotometer (Japan Mollecular Devices Co .; SPECTRAmax250).

2) Preparation of calibration curve

Glucose aqueous solutions of various concentrations were prepared and operated in the same manner as in 1) to prepare a calibration curve for the amount of glucose and glucose-derived carbonyl compounds.

3) Quantification of Carbonyl Compounds

Glucose concentration in the sample solution was measured using a glucose measurement kit (Wakko Pharmaceutical, Glucose CII-Wakko). The amount of carbonyl compounds derived from glucose was calculated | required from the analytical curve. The amount of carbonyl compound derived from glucose was subtracted from the total amount of carbonyl compound in the sample solution to obtain the amount of carbonyl compound in the sample solution.

The results are shown in FIG. By adding 2 mg of carbonyl compound trap beads to the peritoneal dialysis solution and stirring at room temperature for 16 hours, the amount of carbonyl compound was reduced by 55%. In addition, when the amount of added carbonyl compound trap bead was increased to 10 mg, the amount of carbonyl compound further decreased.

From these results, it became clear that the carrier and the carbonyl compound trapping agent were immobilized to suppress the production and / or accumulation of the carbonyl compound in the peritoneal dialysis solution.

Example 8 Trapping of Carbonyl Compounds in Dicarbonyl Solution by Activated Carbon

Activated carbon was used as the carbonyl compound trapping agent to examine the effect of removing the carbonyl compound in the dicarbonyl solution. Into a tube containing 25 µg or 50 µg of activated carbon (wako Pure Chemical Industries, Ltd.), 990 µl of dicarbonyl solution (all 100 µM) in which a dicarbonyl compound was dissolved in a phosphate buffer solution (hereinafter referred to as PBS) was dispensed. As the dicarbonyl compound, glyoxal, methylglyoxal and 3-deoxyglucoson were used. This tube was set to a rotator and stirred for 19 hours at room temperature. After stirring, the solution in the tube was filtered through a centrifugal filtration tube (milipase, UFC30GVO0) having a pore size of 0.22 µm, and the concentration of each dicarbonyl compound was measured using high performance liquid chromatography. The measuring method followed a well-known method.

The results are shown in FIG. When 25 μg of activated carbon was added to 900 μl of dicarbonyl solution, 71% for glyoxal (G0), 94% for methylglyoxal (MGO), and 93% for 3-deoxyglucosone (3DG) Trapped by When 50 µg of activated carbon was used, 85% in glyoxal and 98% in methylglyoxal or 3-deoxyglucoson showed that most of the dicarbonyl compounds tested were trapped by activated carbon.

Example 9 Trapping of Carbonyl Compounds in Peritoneal Dialysis Solution by Activated Carbon

Activated carbon was used as the carbonyl compound trapping agent, and the effect of removing the carbonyl compound in the peritoneal dialysis solution was examined. 900 µl of peritoneal dialysis solution (Baxter Ltd .; Dianeal PD-4, 1.5, trade name) was dispensed into a tube containing 25 µg or 50 µg of activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.). This tube was set to a rotator and stirred for 19 hours at room temperature. After stirring, the peritoneal dialysis solution in the tube was filtered with a centrifugal filtration tube (Mollipoase, UFC30GV00) having a pore size of 0.22 μm, and the concentration of glyoxal, methylglyoxal and 3-deoxyglucoson contained in the filtrate was changed to a high-speed liquid. Measured using chromatography. The measuring method followed a well-known method.

The results are shown in FIG. When 25 μg of activated carbon was added to 900 μl of peritoneal dialysis solution, 56% in glyoxal (GO), 71% in methylglyoxal (MG0), and 3-deoxyglucosone 3DG), 62% was trapped by activated carbon. When 50 µg of activated carbon was used, it was confirmed that 64% of glyoxal, 78% of methylglyoxal, and 77% of 3-deoxyglucoson were trapped by activated carbon.

Example 1 Trapping Effects of Guanidine, Aminoguanidine, and Biguanide Agents on Glyoxal, Methylglyoxal, and 3-Deoxyglucoson

50 µl of a mixed solution of glyoxal, methylglyoxal or 3-deoxyglucose (1 mM each), 400 µl of 0.1 M phosphate buffer (pH 7.4), 50 µl of 30 mM guanidine, aminoguanidine, or biguanide solution It mixed and incubated at 37 degreeC. Metformin, buformin, and phenformin were used for the biguanide agent. After the end of incubation, glyoxal, methylglyoxal and 3-deoxyglucosone were made quinoxaline derivatives using o-phenylenediamine, and the respective concentrations were measured using high performance liquid chromatography.

The results are shown in Fig. 18 (Guanidine), Fig. 19 (Metformin), Fig. 20 (Bupformin), Fig. 21 (Penformin), and Fig. 22 (Aminoguanidine). Guanidine, aminoguanidine and certain biguanides have been found to significantly lower the concentration of methylglyoxal, in particular. In aminoguanidine, the concentration is drastically decreased with respect to methylglyoxal, and also with 3-deoxyglucoson, in which no significant reduction in concentration was found in other biguanides.

Example 11 Trap Effect of SH Agents on Glyoxal, Methyl Glyoxal, and 3-Deoxyglucosone

50 µl of a mixed solution of glyoxal, methylglyoxal and 3-deoxyglucose (1 mM each), 400 µl of 0.1 M phosphate buffer (pH 7.4) and 50 µl of 30 mM SH compound solution were mixed and incubated at 37 ° C. Bait. Cysteine, N-acetylcysteine and GSH were used for the SH compound. After the end of incubation, glyoxal, methylglyoxal and 3-deoxyglucosone were made quinoxaline derivatives using o-phenylenediamine, and the respective concentrations were measured using high performance liquid chromatography.

The results are shown in Fig. 23 (cysteine), Fig. 24 (N-acetylcysteine) and Fig. 25 (GSH). The action of significantly lowering the concentration was confirmed for both glyoxal and methylglyoxal in any SH compound.

EXAMPLE 12 The Trap Effect of Albumin on Glyoxal, Methylglyoxal, and 3-Deoxyglucoson

50 µl of a mixed solution of glyoxal, methylglyoxal, and 3-deoxyglucosone (1 mM each), 400 µl of 0.1 M phosphate buffer (pH 7.4), and 50 µl of bovine serum albumin solution at 10 mg / ml were mixed. Incubated at ° C. After the end of incubation, glyoxal, methylglyoxal and 3-deoxyglucosone were made quinoxaline derivatives using o-phenylenediamine, and the respective concentrations were measured using high performance liquid chromatography.

The results are shown in FIG. Bovine serum albumin has been shown to significantly reduce the concentrations of glyoxal and methylglyoxal.

Example 13 Inhibition of Pentosidine Formation When SH Compound was Added to Peritoneal Dialysis Solution and Incubated at 37 ° C

490 μl of peritoneal dialysis solution (Baxter Ltd .; Dianeal PD-4, 1.5), 70 μl of SH compound dissolved in 0.1 M phosphate buffer (pH 7.4), and bovine serum albumin in peritoneal dialysis solution (Baxter Ltd .; Dianeal PD-4, 140 μl of the solution dissolved in 1.5) to 30 mg / ml was mixed and incubated at 37 ° C. for one week. Cysteine, N-acetylcysteine, GSH, and aminoguanidine were used for the SH compound. After completion of incubation, 50 µl of 10% trichloroacetic acid was added to 50 µl of the solution, and centrifuged to precipitate the protein. The protein was washed with 300 μl 5% trichloroacetic acid and dried. Next, 100 µl of 6N HCl was added, the mixture was heated to 110 ° C for 16 hours, and pentosidine was quantified by high performance liquid chromatography (T. Miyata et al., 1996, J. Am. Soc. Nephrol., 7: 1198-1206, T. Miyata et al., 1996, Proc. Natl. Acad. Sci. USA., 93: 2353-2358).

The results are shown in FIG. By addition of the SH compound, it was confirmed that the amount of pentosidine produced was significantly suppressed.

According to this invention, the obstacle by the carbonyl compound which annoyed the patient in peritoneal dialysis can be removed easily. In the known peritoneal dialysis solution, the peritoneum of the dialysis patient was always under carbonyl stress due to the carbonyl compound leached in vivo by dialysis together with the carbonyl compound produced during the production. In contrast, in the present invention, the carbonyl compound generated in the peritoneal dialysis solution can be effectively removed, thereby contributing to the improvement of carbonyl stress in dialysis patients. Further, by introducing the carbonyl compound trapping agent into the abdominal cavity or circulating the dialysate to the carbonyl compound trap cartridge, the carbonyl compound leached in the abdominal cavity can be effectively inactivated or removed. As such, the present invention provides a very effective approach as a countermeasure against disorders caused by peritoneal dialysis, including peritoneal disorders resulting from carbonyl compounds accompanying peritoneal dialysis.

In addition, in the present invention, since the carbonyl compound derived from the decomposition of glucose during heat sterilization or long-term preservation is eliminated, a peritoneal dialysis solution having a pH of neutral region, which has been difficult to prepare conventionally for the decomposition of glucose, is provided. It is possible to do more physiological peritoneal dialysis.

The peritoneal dialysis solution of the present invention can be carried out only by a simple operation of contact or administration with a carbonyl compound trapping agent, and does not require any special manufacturing equipment. Accordingly, the carbonyl compound trapping agent according to the present invention, the peritoneal dialysis solution using the same, and a method for producing the same, create a new concept of treatment in peritoneal dialysis.

Claims (12)

Intraperitoneal carbonyl stress state improving agent in peritoneal dialysis containing a carbonyl compound trapping agent as an active ingredient. The carbonyl stress state improving agent according to claim 1, wherein the carbonyl compound trapping agent is immobilized on an insoluble carrier. The carbonyl stress state improving agent according to claim 1, wherein the carbonyl compound trapping agent is for incorporation into the peritoneal dialysis solution. The carbonyl compound trapping agent according to any one of claims 1 to 3, wherein the carbonyl compound trapping agent is a compound selected from the group consisting of aminoguanidine, pyridoxamine, hydrazine, biguanide compound, SH group-containing compound, or derivatives thereof. Carbonyl Stress Relievers. The carbonyl stress condition improving agent according to any one of claims 1 to 3, wherein the carbonyl compound trapping agent is a Maillard reaction inhibitor. The carbonyl stress state improving agent according to claim 1, wherein the carbonyl compound trapping agent is an insoluble compound in the peritoneal dialysis solution capable of adsorbing the carbonyl compound. A cartridge for trapping carbonyl compounds in a peritoneal dialysis solution filled with the carbonyl compound trapping agent according to claim 2 and / or 6. A method for preparing a peritoneal dialysis solution, wherein the carbonyl compound content is reduced, including the step of passing the peritoneal dialysis solution to the carbonyl compound trap cartridge of claim 7. a) contacting the carbonyl compound trapping agent according to claim 2 and / or 6 with the peritoneal dialysis solution, and b) separating the carbonyl compound trapping agent and the peritoneal dialysis solution; A method for preparing a peritoneal dialysis solution containing a reduced carbonyl compound content. A peritoneal dialysis solution containing a carbonyl compound trapping agent. The peritoneal dialysis solution according to claim 10, wherein the peritoneal dialysis solution contained in the divided chambers of the first chamber and the second chamber, the reducing sugar is contained in the first chamber, and the carbonyl compound trapping agent is contained in the second chamber. The peritoneal dialysis solution according to claim 10, wherein the carbonyl compound trapping agent is for administration intraperitoneally with the peritoneal dialysis solution.